Chromoplasts are colored plastids that synthesize and store massive amounts of carotenoids.Chromoplast number and size define the sink strength for carotenoid accumulation in plants.However,nothing is known about the mechanisms controlling chromoplast number.Previously,a natural allele of Orange (OR),ORHis,was found to promote carotenoid accumulation by activating chromoplast differentiation and increasing carotenoid biosynthesis,but cells in orange tissues in melon fruit and cauliflower OR mutant have only one or two enlarged chromoplasts.In this study,we investigated an ORHis variant of Arabidopsis OR,genetically mimicking the melon ORHis allele,and found that it also constrains chromoplast number in Arabidopsis calli.Both in vitro and in vivo experiments demonstrate that ORHis specifically interacts with the Membrane Occupation and Recognition Nexus domain of ACCUMULATION AND REPLICATION OF CHLOROPLASTS 3 (ARC3),a crucial regulator of chloroplast division.We further showed that ORHis interferes with the interaction between ARC3 and PARALOG OF ARC6 (PARC6),another key regulator of chloroplast division,suggesting a role of ORHis in competing with PARC6 for binding to ARC3 to restrict chromoplast number.Overexpression or knockout of ARC3 in Arabidopsis ORHis plants significantly alters total carotenoid levels.Moreover,overexpression of the plastid division factor PLASTID DIVISION 1 greatly enhances carotenoid accumulation.These division factors likely alter carotenoid levels via their influence on chromoplast number and/or size.Taken together,our findings provide novel mechanistic insights into the machinery controlling chromoplast number and highlight a potential new strategy for enhancing carotenoid accumulation and nutritional value in food crops.